Hematological malignancies are a major public health problem. It has been estimated that in the year 2000, more than 50,000 new cases of non-Hodgkin's lymphoma and more than 30,000 new cases of leukemia occurred in the United States (Greenlee, R. T. et al., CA Cancer J. Clin., 50:7-33 (2000)) and more than 45,000 deaths were expected from these diseases. Many more patients live with chronic disease-related morbidity. Unfortunately, in a high percentage of patients, conventional therapies are not able to induce long term complete remissions.
In the past several years immunotoxins have been developed as an alternative therapeutic approach to treat these malignancies. Immunotoxins were originally composed of an antibody chemically conjugated to a plant or a bacterial toxin. The antibody binds to the antigen expressed on the target cell and the toxin is internalized causing cell death by arresting protein synthesis and inducing apoptosis (Brinkmann, U., Mol. Med. Today, 2:439-446 (1996)).
Hematological malignancies are an attractive target for immunotoxin therapies because tumor cells are easily accessible and the target antigens are highly expressed (Kreitman, R. J. and Pastan, I., Semin. Cancer Biol., 6:297-306 (1995)). One of these antigens is CD25. A clinical trial with immunotoxin LMB-2 (anti-Tac(Fv)PE38) that targets CD25 showed that the agent was well tolerated and that it had substantial anti-tumor activity (Kreitman, R. J. et al., Blood, 94:3340-3348 (1999); Kreitman, R. J. et al., J. Clin. Oncol., 18:16222-1636 (2000)). A complete response was observed in one patient with Hairy Cell Leukemia and partial responses were observed in patients with Hairy Cell Leukemia, chronic lymphocytic leukemia, cutaneous T cell lymphoma, Hodgkins disease and adult T cell leukemia.
Another antigen that has been used as an immunotoxin target is CD22, a lineage-restricted B cell antigen expressed in 60-70% of B cell lymphomas and leukemias. CD22 is not present on the cell surface in the early stages of B cell development and is not expressed on stem cells (Tedder, T. F. et al., Annu. Rev. Immunol., 5:481-504 (1997)). Clinical trials have been conducted with an immunotoxin containing an anti-CD22 antibody, RFB4, or its Fab fragment, coupled to deglycosylated ricin A. In these trials, substantial clinical responses have been observed; however, severe and in certain cases fatal, vascular leak syndrome was dose limiting (Sausville, E. A. et al., Blood, 85:3457-3465 (1995); Amlot, P. L. et al., Blood, 82:2624-2633 (1993); Vitetta, E. S. et al., Cancer Res., 51:4052-4058 (1991)).
As an alternative approach, the RFB4 antibody was used to make a recombinant immunotoxin in which the Fv fragment in a single chain form is fused to a 38 kDa truncated form of Pseudomonas exotoxin A (PE38). PE38 contains the translocating and ADP ribosylating domains of PE but not the cell-binding portion (Hwang, J. et al., Cell, 48:129-136 (1987)). RFB4 (Fv)-PE38 is cytotoxic towards CD22-positive cells (Mansfield, E. et al., Biochem. Soc. Trans., 25:709-714 (1997)). To stabilize the single chain Fv immunotoxin and to make it more suitable for clinical development, cysteine residues were engineered into framework regions, mutating arginine residue 44 of the VH to cysteine and mutating glycine residue 100 of the VL to cysteine (Mansfield, E. et al., Blood, 90:2020-2026 (1997)), the positions numbered according to the Kabat Numbering System, generating the molecule RFB4 (dsFv)-PE38.
RFB4 (dsFv)-PE38 is able to kill leukemic cells from patients and induced complete remissions in mice bearing lymphoma xenografts (Kreitman, R. J. et al., Clin. Cancer Res., 6:1476-1487 (2000); Kreitman, R. J. et al., Int. J. Cancer, 81:148-155 (1999)). RFB4 (dsFv)-PE38 (BL22) is currently being evaluated in a phase I clinical trial at the National Cancer Institute in patients with hematological malignancies. Sixteen patients with purine analogue resistant hairy cell leukemia were treated with BL22 and 11 (86%) have achieved complete remissions (Kreitman, R. J. et al., N. Engl. J. Med. (2001)).
Because of the clinical benefits obtained with BL22, and because improved binding affinity has been shown to improve selective tumor delivery of scFvs (Adams et al., Cancer Res. 58:485-490 (1998)), improving the binding affinity of scFvs and other targeting moieties (such as dsFvs, Fabs. and F(ab′)2) of immunoconjugates could improve the efficiency of these agents in delivering effector molecules to malignant B-cells. Improved targeting would likely decrease the dose necessary to achieve complete remission of these cancers.
The factors that influence binding affinity are multifaceted and obtaining mutant scFvs with improved affinity is not trivial. Although antibody-antigen crystal structure can suggest which residues are involved in binding, but atomic resolution structural data are not available for most antibodies. Moreover, even when such data is available it cannot generally be predicted which residues and which mutations will result in an antibody with increased antigen binding activity.